Method for the detection of fluoride or hydrogen fluoride and detection kit

ABSTRACT

The present invention relates to a method for detecting and/or measuring the concentration of fluoride (F − ) or hydrogen fluoride (HF) in a sample, comprising the steps consisting of bringing said sample, in aqueous solution, into contact with a silylated organic compound in order to obtain a measurement solution, with said silylated organic compound being desilylated when it is in the presence of hydrofluoric acid or a fluoride, with the silylated organic compound and the desilylated organic compound being able to be detected and/or measured separately from each other; and detecting and/or measuring, in said measurement solution, the appearance of the desilylated against compound or the disappearance of the silylated organic compound, which takes place if fluoride or hydrogen fluoride is present in the sample. The method enables the presence of hydrogen fluoride or of fluorine to be detected very easily and expediently at concentrations of 10 −2  l of HF/10 6  l of air (10 ppb) or else of 0.5 to 1 μg/ml of HF in solution. The kit of the present invention comprises the components which are required for implementing this method. The method of the invention makes it possible to detect fluorine at concentrations of the order of 0.001 μg/ml.

TECHNICAL FIELD

The present invention relates to a method for detecting and/or measuringthe concentration of fluoride (F⁻) or hydrogen fluoride (HF) which ispresent in a sample and to a detection kit for implementing this method.This invention makes it possible to measure an environmental pollutantefficiently and sensitively. The invention is based on a method which isentirely original and is easy to implement.

Hydrogen fluoride is a strong inorganic acid which is colorless and verysoluble in water, where it forms hydrofluoric acid. HF is a gas which iswidely used in industry, in particular for producing polymers, coolingliquids and fire-extinguishing products, for refining aluminum, forpreparing nuclear fuels and for manufacturing components forelectronics. In addition, it is a product which is emitted during thecombustion of coal, household or industrial wastes and plastics.

HF is toxic above concentrations of the order of 3×10⁻² l of HF/10⁶ l ofair (30 ppm). HF is a powerful irritant which can cause burns, resultingfrom exposure of the skin/mucous membranes, as well as inflammation ofthe upper and lower airways. In addition, its absorption can give riseto metabolic disturbances.

Furthermore, this product is very corrosive in regard to a large numberof materials such as iron, bronze and glass.

The protection of people, of the environment and of industrial equipmentconsequently makes it necessary to set up means for monitoring itsconcentration, particularly in industrial effluents and in laboratories,as well as in the atmosphere surrounding these installations, in orderto be able to take appropriate measures if dangerous concentrations ofHF are detected.

Norms for acceptable atmospheric concentrations of HF have beenestablished in a large number of countries. These norms are generallybetween 5×10⁻⁵ and 5×10⁻⁴ l of HF/10⁶ l of air (between 0.05 and 0.5ppm).

In the remainder of the description, the references between squarebrackets [ ] refer to the reference list which is appended hereto.

PRIOR ART

To date, a large number of methods have been developed, and described,for measuring HF or fluoride ions. In general, these techniques arebased on chemical, electrochemical, spectrometric or optical detection.A large number of devices which can be used for detecting and measuringthe quantity of HF are available commercially.

Document [1] in the appended reference list describes a number ofelectrochemical, spectrophotometric and chromatographic methods whichcan be used for detecting and measuring the quantity of HF in a sample.

A reference method, termed ID-110, which is based on using specificelectrodes is described on the OSHA website with the reference number[2]. While the sensitivity of this method is 1.2×10⁻² l of HF/10⁶ l ofair (12 ppb), the method is a laboratory test which is difficult toapply in the field. As far as field methods are concerned, they are muchless sensitive and of the order of 0.2 l of HF/10⁶ l of air (200 ppb).

Another method uses a detector which is based on a silicon carbidesubstrate (“Metal Insulator Semiconductor”: MIS). This method isdescribed, for example, in the document with the reference number [3].Various manufacturers propose a variety of electronic detectioninstruments for measuring HF. Examples of these are the OEM FluorineSensor (trade mark) instrument from the Bionics Instrument company andwhich is described on the website with the reference. [4], or theLaserGas (trade mark) instrument from the NORSK ELEKTRO OPTIKK A/Scompany.

The detection limits which are given for these instruments are expressedin parts per million or parts per billion (ppm or ppb) in the case of agaseous medium or as nanograms or micrograms per milliliter (ng/ml orμg/ml) in the case of an aqueous medium. In general, the limits are ofthe order of 10⁻⁴ l of HF/10⁶ l of air (0.1 ppm) or, when in solution,of from 1 to 1000 ng of HF/ml in the case of the best systems.

Unfortunately, these methods and instruments require space, aredifficult to move about, are sometimes difficult to implement and arefrequently expensive.

A “ideal means for detecting” HF should combine the following features:

-   -   sensitivity: measurement of at least 10⁻⁴ l of HF/10⁶ l of air        (0.1 ppm),    -   cost: this should not be too high in order to make it possible,        if necessary, to readily increase the number of detectors,    -   availability: a number of published techniques are not available        on the market, particularly because of the difficulty in        implementing them,    -   mobility: it should be possible to easily move the means, in        order to be able to use it at different sites,    -   rapidity of implementation, and of the use of the results.

It appears that none of the methods or devices of the prior art combinesall these properties.

There is therefore a real need for novel techniques which combine asmany as possible of the above mentioned features of the “ideal detectionmeans”.

ACCOUNT OF THE INVENTION

The object which is achieved by the present invention is specificallythat of supplying a method and a kit for detecting HF, which method andkit possess all the abovementioned features and furthermore do notsuffer from the abovementioned drawbacks of the methods and devices ofthe prior art.

This object is achieved by means of a method for detecting and/ormeasuring the concentration of fluoride (F⁻) or hydrogen fluoride (HF)in a sample, which method comprises the following steps:

-   -   bringing said sample, in aqueous solution, into contact with a        silylated organic compound in order to obtain a measurement        solution, with said silylated organic compound being desilylated        when it is in the presence of hydrofluoric acid or of fluoride,        with the silylated organic compound and the desilylated organic        compound being able to be detected and/or measured separately        from each other; and    -   detecting and/or measuring, in said measurement solution, the        appearance of the desilylated organic compound, or the        disappearance of the silylated organic compound, which takes        place if fluoride or hydrogen fluoride is present in the sample.

In the description which follows, fluoride (F⁻), hydrogen fluoride (HF)and hydrofluoric acid are implicitly designated by the terms “fluorine”or “fluorine and its derivatives”. Fluoride(s) is understood as meaningsalts of fluorine.

Fluorine is a very nucleophilic atom and, for this reason, it canintervene in nucleophilic substitution reactions. More particularly, itcan specifically attack bonds of the silicon-oxygen (Si—O) type in themanner shown in the following chemical equation.

silylated compound R⁴ hydrogen fluoride silyl group compound R⁴(desilylated)

General Principle of the Reaction of HF on the Si—O Bond (Desilylation)

In this chemical equation, R¹, R² and R³ are substituents of Si andform, with the latter, a silylation group of the organic compound R⁴within the meaning of the present invention.

R¹, R² and R³ can be selected independently from C₁ to C₆ alkyls. Forexample, R¹, R² and R³ can be selected independently from the groupconsisting of methyl, ethyl, propyl and butyl.

In this chemical equation, —R⁴ therefore represents the organic compoundwhich is silylated (on the left) or desilylated (on the right) duringthe implementation of the method of the invention. In order to be ableto be silylated, this compound contains at least one hydroxyl functionwhich is accessible for silylation. This compound is advantageously anorganic compound in order to facilitate its detection when the method ofthe invention is implemented. In general, it has a molecular weight offrom 250 to 200 000 g.mol⁻¹, for example from 250 to 1500 g.mol⁻¹, inparticular for issues of solubility, and therefore sensitivity, andreproducibility of the fluorine detection and/or measurement results.The compound can be a hydroxylated compound which is selected, forexample, from estradiol, peptides, for example peptides containing from3 to 50 amino acid residues, homovanillic acid, amphotericin, steroids,cytokines and arachidonic acid, or derivatives of these compounds.

A peptide which can be used in the present invention is described, forexample, in reference [5]; homovanillic acid which can be used, andderivatives thereof, are described, for example, in references [6] and[7]; amphotericin which can be used is described, for example, inreferences [8] to [10]; steroids and steroid derivatives which can beused in the present invention are described, for example, in references[11] to [17]; cytokines and cytokine derivatives which can be used inthe present invention are described, for example, in references [18] to[20]; and arachidonic acid and its derivatives which can be used aredescribed, for example, in references [21] to [24].

The essential thing is that the silylated organic compound and thedesilylated organic compound can be detected and/or measured separatelyfrom each other in order to make it possible to detect and/or measurethe disappearance of one, and/or the appearance of the other, of thesecompounds in the measurement solution. Thus, the method of the inventionis based on detecting the silylated organic compound or the desilylatedorganic compound, or these two compounds, but then separately from eachother.

According to the invention, any suitable reagent can be used forattaching a silyl group (silylation) to the selected organic compound,provided that the desilylated organic compound, or native form of theorganic compound, can be recovered intact by the simple action offluorine, or its derivatives, on the silylated organic compound. Thesilyl groups or functions which can be used are described above in thechemical equation.

Mention may be made, by way of example, of the following reagents whichcan be used for silylating hydroxylated organic compounds for thepurpose of implementing the present invention:N,O-bis(trimethylsilyl)trifluoroacetamide (or BTSFA),N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (or MTBSTFA),trimethylsilyl (or TMS), tert-butyldimethylsilyl (or t-BDMS),N,O-bis(trimethylsilyl)acetamide (or BSA), hexamethyldisilazane (orHMDS), N-methyltrimethylsilyl-trifluoroacetamide (or MSTFA),trimethylchlorosilane (or TMCS), trimethylsilylimidazole (or TMSI), etc.

Other reagents and operational modes for silylating which can be usedfor implementing the present invention can also be found, for example,in references [25] to [30] in the appended reference list.

In general, these reagents are simple to use and readily available andmake it possible to transfer a silyl function (containing a siliconatom) onto a hydroxyl function belonging to the selected organiccompound.

Mention may be made, in particular, of the following two reagents forthe purpose of illustrating the fact that the inventors haveadditionally, in the present invention, demonstrated a variation in thesensitivity and selectivity of the detection and/or measurement offluorine in accordance with the invention in dependence on the silylgroup which is used:

-   -   BTSFA, which attaches a trimethylsilyl (—Si(CH₃)₃) function to        the —OH groups of the selected organic compound;    -   MTBSTFA, which attaches a dimethyl tert-butylsilyl        (Si(CH₃)₂C(CH₃)₃ function to the —OH groups of the selected        organic compound.

BTSFA exhibits the advantage of transferring, to the hydroxylatedcompounds, a silyl which is not particularly hydrophobic, therebyenabling the silylated organic compound to have relatively goodsolubility. However, the silylated compounds which are obtained do notalways exhibit specificity toward HF in regard to their conversion intodesilylated compounds when the method of the invention is implemented.They can also be converted into desilylated compounds by other acidswhich are present in the sample. This reagent can therefore preferablybe used for detecting fluorine when specificity in regard to thefluorine is not required or else for detecting fluorine in samples inwhich the concentration of the other acids is sufficiently low as not tointerfere with the detection or the measurement of the fluorine.

MTBSTFA transfers a more hydrophobic silyl group to the hydroxylatedcompounds and the solubility of the resulting silylated compounds inaqueous medium is less good than in the case of the silylated compoundswhich are obtained with BTSFA. On the other hand, and quiteunexpectedly, the desilylation of the compounds which have beensilylated by MTBSTFA is more specific for HF than for the other acidstested, as is demonstrated in the examples. This reagent can thereforeadvantageously be employed for detecting hydrogen fluoride in a samplewhen specificity in regard to fluorine is required, for example when thesample contains other acids at concentrations significant.

Thus, the inventors have discovered, in particular by using thesereagents, that the specificity of the attack on the Si—O bond by thefluorine also depends on the radicals which are attached to the siliconand oxygen atoms. Thus, when the silylation group is selectedappropriately, the other inorganic or organic acids and salts in thesample do not interfere with the measurements which are taken when themethod of the invention is implemented. For example, HF has almost thesame desilylation activity as HCl when the method of the invention isimplemented using —Si—(CH₃)₃ and an activity which is about 1000 timesgreater (in the case of HF as compared to HCl) when the method of theinvention is implemented using —O—Si—(CH₃)₂—C(CH₃)₃.

As the above chemical equation shows, the conversion of the silylatedorganic compound into a desilylated organic compound by the fluorine orits derivatives is proportional to the quantity of HF which is presentin the sample. It is therefore possible, by means of the method of theinvention, to detect and measure, at one and the same time, the fluorineand its derivatives which are present in a sample. The essential featureof the invention, and its inventive nature, consist precisely indetecting the fluoride and/or the hydrogen fluoride by using a detectionmeans which measures the difference between the silylated organiccompound and the unsilylated (desilylated) organic compound, andtherefore detecting/quantifying the desilylation which is catalyzed bythe presence of the fluorine.

The step of bringing into contact can be effected by mixing the sampleand the silylated organic compound in an aqueous solution. The mixingcan be effected by simply adding the sample and the fluorine in solutionor by means of active mixing, for example using a mechanical or magneticstirrer, by means of ultrasonication, etc., of the sample and thefluorine in solution, with the objective naturally being that ofpromoting the interaction between the fluorine, if it is present, andthe silylated organic compound so as to ensure that they react together.

The sample can be a liquid, solid or gaseous sample. When the sample isnot liquid, the step of bringing the sample and the silylated organiccompound of the method of the invention into contact in aqueous solutionnaturally comprises dissolving the sample in an aqueous solution, forexample by means of bubbling when the sample is gaseous or by means ofmixing or solubilizing while stirring when the sample is solid, with theaim of this dissolution being that of once again finding the fluorine ofthe gaseous or solid sample in the contacting aqueous solution. Themethods which are used for this dissolution are well known to theskilled person.

The inventors have demonstrated that the addition of a water-miscibleorganic solvent to said contacting aqueous solution can substantiallyincrease the sensitivity of the detection and/or measurement of thefluorine by the method of the invention. By way of example, the organicsolvent can be selected from dimethylformamide (DMF), dimethyl sulfoxide(DMSO), ethanol or methanol or equivalent organic solvents which areknown to the skilled person. Thus, the inventors have measured anincrease in the sensitivity of the detection of the fluorine by a factorof 100, and even of 500, when such solvents are used. For example, whenusing DMSO, the sensitivity of detection extends to 0.001 μg/ml.

The organic solvent could have several different effects: facilitatingthe solubility of the silylated organic compound, facilitating thedesilylation or decreasing interference in the detection (noticed, inparticular, in the immunotests). Whatever it may be, one of theseeffects alone is not sufficient to explain the quite surprisinglysignificant increase in the sensitivity of detection in the presence ofthe organic solvent. This organic solvent can be present in a quantityin the range from 1 to 99% by volume of the contacting aqueous solution,advantageously from 50 to 95% by volume of the contacting aqueoussolution, with the remainder being water.

The origin of this solvent can advantageously be the recovery solvent ofthe silylated organic compound after it has been prepared, that is tosay after the organic compound has been silylated. The step of bringinginto contact can then be effected by mixing the aqueous solution of thefluorine and the organic solution of the silylated organic compound.Otherwise, the organic solvent can be added independently to thecontacting solution.

The detection of the silylated organic compound which disappears or ofthe desilylated organic compound which appears can be effected using anymeans known to the skilled person which makes it possible to demonstratethe presence of one or other of these compounds separately. The meanscan, for example, be a detection and/or a measurement which is effectedby gas chromatography or a detection and/or a measurement which iseffected by means of an immunological test.

For the purpose of carrying out the measurement, a standard series can,in a general manner, be first of all determined by applying the methodof the invention using known quantities of hydrofluoric acid orfluorine, or using known quantities of the silylated or unsilylatedorganic compound. This standard series will then make it possible, bymeans of simple extrapolation, to determine the quantity of fluorine, ofhydrogen fluoride or of hydrofluoric acid which is present in thesample.

In a first embodiment of the present invention, the detection and/or themeasurement can be effected by means of gas chromatography. This isbecause this technique makes it possible to separately detect and/ormeasure the silylated form or the desilylated form of the organiccompound according to the invention and therefore the fluorine if it ispresent. The chromatographic techniques which can be used are thosewhich are known to the skilled person. The techniques described inreferences [31] to [33] in the appended reference list may be mentionedby way of example.

The organic compounds which can be used in this first embodiment can bethe abovementioned hydroxylated organic compounds. The silylationreagents and techniques can also be those which are mentioned above.

In a second embodiment of the present invention, the detection and/ormeasurement of the appearance of the unsilylated organic compound or ofthe disappearance of the silylated organic compound is advantageouslyeffected using an immunological test, that is to say using antibodieswhich are directed either against the unsilylated organic compound oragainst the silylated organic compound. No approach of this type isdisclosed in the prior art. In addition, this embodiment makes itpossible to detect the fluorine much more sensitively than do thetechniques of the prior art.

In a general manner, antibodies are proteins which are able, with verygreat specificity, to recognize, and bind to, structures termed antigensin order to form a detectable antibody/antigen complex. However, sinceHF is a molecule of very small size, it is impossible to produceantibodies against it. Since they were unable to produce antibodieswhich are directed directly against HF, the inventors adopted anoriginal strategy based on the abovementioned chemical properties of thefluorine and on using particular silylated organic compounds whichexhibit the special feature of giving rise to the generation ofantibodies when they are injected into an animal.

The organic compounds which can be used in this second embodiment areorganic compounds which possess one or more hydroxyl group(s) whichenable a silyl group within the meaning of the present invention to beattached and which induce, in an animal into which they have beeninjected, the specific production of antibodies which are directedagainst the silylated organic compound or against the unsilylatedorganic compound. The organic compounds generally have a molecularweight of from 250 to 200 000 g.mol⁻¹, for example from 250 to 1500g.mol⁻¹. The organic compound can, for example, be an organic compoundwhich is selected from the above-mentioned hydroxylated organiccompounds, for example selected from estradiol; peptides, for examplepeptides comprising from 3 to 50 amino acid residues, for example thetetrapeptide acetylated Ser-Asp-Lys-Pro (AcSDKP), which is mentioned inreference [5]; homovanillic acid; amphotericin; steroids; cytokines;arachidonic acid; or derivatives of these compounds. The organiccompounds can, for example, be compounds such as those mentioned inreferences [5] to [24].

As an illustrative example, it is possible to use estradiol or itsderivatives. According to the invention, “estradiol” is understood asmeaning, for example, estratiene-1,3,5 diol-3,17μ or 17μ-diol, or theirderivatives, or other, equivalent, compounds, provided they are able togenerate the formation of antibodies when they are injected into ananimal, for example a mouse, and provided they are able to be silylatedwithin the meaning of the present invention.

Examples of silylated estradiols which have been obtained by theinventors and which can be used in the present invention are depictedbelow. Estradiol possesses —OH functions to which it is possible toattach a silyl group by means of Si—O bonds, as depicted below (to becompared, for example, to native estradiol (Es), which is depicted inthe appended FIG. 1).

The reagents which can be used for silylating the organic compound whichis selected for implementing this second embodiment of the presentinvention can be those mentioned above, for example BTSFA or MTBSTFA.

The antibodies which can be used in this embodiment of the inventionexhibit the special feature of recognizing the silylated organiccompound and the unsilylated organic compound differently. Theseantibodies can be monoclonal. They can be prepared by means of thetechniques which are customary for preparing antibodies of this type,for example by injecting the silylated organic compound, or else theunsilylated organic compound, into a mouse in order to obtain a mouseantibody which is specific for only one of these compounds. Tests can becarried out on different batches of prepared antibodies in order toselect antibodies which are specific for only one of the two compounds(silylated or unsilylated) for the purpose of implementing the method ofthe invention. The skilled person is familiar with these techniques forpreparing antibodies.

Examples of Silylated Estradiols which can be Used in the PresentInvention

By way of example, the documents with the reference numbers [34] to [38]outline appropriate methods for preparing antibodies which can be usedfor implementing the present invention.

Also by way of example, references [5] to [24] and [39] to [42]additionally describe antibodies which can be used for implementing thepresent invention when the organic compound is a silylatable compoundwhich is selected from the above list.

In the example which is illustrated by the appended FIG. 1, the antibodywhich is directed against estradiol and which is used in the immunoassayis selected such that it no longer recognizes estradiol, or recognizesit much less well (i.e. in a manner which is sufficient for thisdifference in recognition to be detected) when the estradiol is modifiedwith a silyl group (also termed “modified estradiol” below). The effectof the fluorine on the modified estradiol is to attack the Si—O bond(desilylation reaction), thereby enabling the native estradiol toreappear, it then being possible for native estradiol to be recognizedby its specific antibody, which is termed “first antibody” below.

Any means for detecting an antigen/antibody recognition which are knownto the skilled person can be used in the present invention for detectingeither recognition of the desilylated organic compound (“native organiccompound”) by its antibody or recognition of the silylated organiccompound by its antibody. This is the reason why the terms “means fordetecting an interaction between a first antibody and the unsilylatedorganic compound” or “means for detecting an interaction between a firstantibody and the silylated organic compound” are used herein. Theoperational conditions for such detections using antibodies are known tothe skilled person. Protocols, reagents, buffers and operationalconditions which can be used are described, for example, in thedocuments with the reference numbers [5] to [24] and [39] to [42].

The detection in accordance with this embodiment can be effected bycarrying out an immunoassay of the “competitive” type or of the“noncompetitive” type, for example such as those which are customarilyemployed in immunological methods of detection and/or immunologicalmethods of measurement.

According to a first variant of this second embodiment of the presentinvention, the detection means can, for example, be a labeled moleculewhich is termed a “tracer” and which enters into competition with thesilylated or unsilylated organic compound, for example silylated orunsilylated estradiol, in regard to said first antibody. This labeledmolecule can use an enzyme, for example acetylcholine esterase, afluorescent label, a luminescent label or a radioisotope for thedetection.

The immunoassay (or immunological measurement) of the silylated orunsilylated organic compound is then said to be “competitive”: it makesuse of an antibody which is directed against the silylated orunsilylated organic compound, for example silylated or unsilylatedestradiol, which is present in a standard series or in a sample, and atracer which is chemically similar to the organic compound according tothe invention, for example estradiol, and which carries a signal(label), for example an enzymic, fluorescent, luminescent or radioactivesignal. In the abovementioned example of estradiol, the tracer can beobtained, for example, by coupling the estradiol to an enzyme, forexample acetylchloline esterase.

A “competitive” detection which can be used for implementing the methodof the invention is depicted diagrammatically in FIGS. 2 and 3. Theorganic compound which is used in these figures is estradiol. Thelabeled molecule or tracer is given the reference “T”, while unsilylated(unmodified) estradiol is given the reference “Es” and silylated(modified) estradiol is given the reference “Es-M”. The first antibodyis, for example, a mouse antibody (sAb). FIG. 2 shows that, whilemodified estradiol (Es-M) cannot be recognized by the antibody (sAb)(this inability is represented by the cross), it is converted, when HFis present, into native estradiol (Es) which can then be recognized bythe first antibody (sAb). It then enters into competition with thetracer (T) (labeled estradiol). The presence of HF can therefore bedetected. In this method, the quantity of tracer which is bound to theantibody is inversely proportional to the quantity of estradiol which ispresent in the sample.

In this method of detection by competition, the means for detecting theinteraction between the first antibody (sAb) and the unsilylated organiccompound (or the means for detecting an interaction between the firstantibody and the silylated organic compound) can comprise a secondantibody (gAb). For example, this second antibody can be a goat antibody(gAb) or a rabbit antibody which is directed against the above-mentionedmouse antibody (sAb) (first antibody). A diagrammatic depiction of thisdetection using a second antibody has been added to the appended FIG. 2.This second antibody (gAb) can be bound to the bottom of a receptacle(R), for example a microtitration plate.

In this example, therefore, the assay consists in effecting acompetition, between the tracer and the estradiol to be measured, inregard to a limited number of antibody molecules. At the end of thereaction, and after the tracer which is not bound to the antibodies hasbeen removed, the signal carried by the antibody-bound tracer can bemeasured.

When the tracer comprises an enzyme, for example acetylcholine esterase(shown diagrammatically by the white square in FIG. 2), the detectionmeans can additionally comprise an enzymic indicator. In theabovementioned example of using acetylcholine esterase, the enzymicindicator can consist of a mixture of acetylthiocholine anddithionitrofluorobenzene. This indicator is transformed into a yellowproduct which is visible to the naked eye or which can be quantifiedusing a spectrophotomer at 414 nm. This indicator is in fact used forapplying the Ellman colorimetric detection and measurement method forimplementing the present invention. This method is described, forexample, in the document with the reference number [43].

According to a second variant of this second embodiment of the presentinvention, the detection and/or measurement of the fluorine inaccordance with the method of the invention is effected using animmunoassay of the “non-competitive” type. As compared to thecompetitive assay, the difficulty in the non-competitive approach islinked to the selected organic compound, which should possess twochemical properties: the possibility of being coupled to a solid phaseand the possibility of being silylated. In addition, having been coupledto the solid phase, the organic compound should be able to bedesilylated under conditions which are as advantageous as in liquidphase. This variant can be applied to the abovementioned hydroxylatedorganic compounds. For example, it can be applied to estradiolderivatives such as those mentioned above, for example estradiol3-carboxymethyl ether, or to peptides, for example the abovementionedAcSDKP.

In this variant, the silylated compound is coupled to a solid phase, forexample a support, for example a microtitration plate such as thosewhich are currently used in laboratories. The sample can be brought intocontact with the silylated compound which is thus immobilized on theplate and the detection and/or measurement can be effected in situ.

The silylated compound can be immobilized on, or coupled to, the supportby any appropriate technique known to the skilled person for attachingone of the silylated organic compounds as defined above to a supportwhile at the same time preserving the reactivity of the silylatedcompound with fluoride ions. Everything depends on the chemical natureof the support and of the silylated organic compound which is selectedfor implementing the present invention. This attachment can be effected,for example, by means of a covalent bond. The support can be afunctionalized support, that is a support to which chemical groupsfacilitating the attachment of the silylated organic compound have beengrafted. The support can, for example, be a plate which comprises aminogroups, for example a Nunc-NH₂ plate, or a plate which is activated withpolylysine, for example when the selected silylated organic compound isan estradiol derivative such as those mentioned above, for exampleestradiol 3-carboxymethyl ether, or a peptide, for example AcSDKP.

Following reaction with the fluoride ions, if they are present in thesample, the silylated compound which is attached to the support istransformed into an unsilylated compound. The detection and/ormeasurement can then be effected by means of an immunoassay technique,for example in the presence of a first antibody which specificallyrecognizes the unsilylated compound.

The recognition of the unsilylated compound by the first antibody can bedemonstrated by any means known to the skilled person, for example bylabeling the first antibody, but also by means of using a secondantibody which recognizes the first antibody and which carries a label(the second antibody is then a tracer). The label can, for example, beone of those which are customarily used by the skilled person, forexample an enzymic signal. For example, the second antibody can be agoat or rabbit antibody (gAb) which is directed against the firstantibody, which can be a mouse antibody (sAb).

One method of “non-competitive” detection is depicted diagrammaticallyin FIG. 18. The organic compound which is used in this figure isestradiol carboxymethyl ether (EstCME). The first of the antibodiesemployed is an anti-estradiol antibody (for example mouse antibody)which is given the reference “sAb” (tracer). The unsilylated(unmodified) estradiol is given the reference “Es” and the silylated(modified) estradiol is given the reference “Es-M”. A second goat orrabbit antibody (gAb), which is directed against the mouse antibody(sAb), is added, for example to the surface of a microtitration plate(R). The gAb antibody is attached to a label, for example an enzyme, inorder to form the tracer “T”. This figure shows that the antibody (sAb)is unable to recognize the modified estradiol (Es-M) (this inability isrepresented by the cross); however, when HF is present, Es-M isconverted into native estradiol (Es) which can then be recognized by thefirst antibody (sAb). The presence of HF can therefore be detected. Thequantity of tracer (gAb+label) bound to the antibody (sAb) is in thiscase proportional to the quantity of desilylated estradiol which ispresent in the sample.

The inventors have determined the operating conditions which areadvantageous for implementing the method of the present invention,whichever embodiment of the invention is employed. These conditions cannaturally be adapted or modified, if necessary, for other silyl groupsemployed, for other silylated organic compounds or for other means ofdetection within the meaning of the present invention. These conditionsare particularly advantageous for the second embodiment of the presentinvention.

Thus, the step of bringing into contact in the method of the inventioncan advantageously be effected at a temperature of from 54 to 64° C.This is because phenomena involving the spontaneous desilylation of thesilylated organic compound can appear at temperatures which are toohigh. The skilled person will be able, without difficulty, to adapt thetemperature at which the method of the invention is implemented independence on the organic compound and the detection method which areselected.

Also advantageously, the bringing into contact in the method of theinvention can be effected at a pH of 4.5 to 6.5, preferably at a pH of5.5, for example in a 50 mM phosphate buffer or using any appropriatebuffer.

This is because phenomena involving the spontaneous desilylation of thesilylated organic compound can appear at pH values which are too acid.In this case, too, the skilled person will be able, without difficulty,to adapt the pH at which the method of the invention is implemented independence on the organic compound and the detection method which areselected.

According to the invention, the concentration of the silylated organiccompound, for example such as estradiol or another compound ofequivalent molecular weight, in the measurement solution can be adaptedin dependence on the molecular weight of the organic compound and on thedetection and/or measurement means employed. In general, theconcentration can be from 1 to 2000 ng/ml when the method of theinvention is implemented, for example from 2 to 500 ng/ml.

The present invention also relates to a kit for implementing the methodof the invention, with said kit comprising the following reagents: asilylated organic compound which is desilylated when it is in thepresence of fluorine or hydrofluoric acid; and a means for detecting, inaqueous solution, the appearance of the desilylated organic compound orthe disappearance of the silylated organic compound.

The silylated organic compound which is desilylated when it is in thepresence of fluorine or hydrofluoric acid is as defined herein.

The means for detecting and/or measuring the appearance of thedesilylated organic compound, or the disappearance of the silylatedorganic compound, in aqueous solution naturally depends on the detectionand/or measurement method which is used for implementing the method ofthe invention. The methods which can be used are as defined above. Thedetection and/or measurement means can therefore comprise one or more ofthe following components: colored indicators, markers such as thosementioned above, enzymes, measurement means such as those mentionedabove, antibodies which are required for detecting and/or measuring thesilylated and/or unsilylated organic compound(s), etc.

For example, the kit can comprise reagents and antibodies which arerequired for implementing an immunoassay of the competitive type, orreagents and antibodies which are required for implementing animmunoassay of the non-competitive type. The kit can comprise one ormore antibodies, for example mouse antibodies, which is/are directedagainst the unsilylated or desilylated organic compound or against thesilylated organic compound. It can additionally comprise one or moreantibod(ies), for example goat or rabbit antibodies, which is/aredirected against the abovementioned antibodies. It can also comprise atracer which makes it possible to demonstrate the immunological reactionwhich has been implemented.

According to the invention, the kit can additionally comprise a supportfor receiving the reagents, for example a polystyrene strip in which oneor more wells have been formed, with this/these well(s) being used as(a) receptacle(s) for the step of bringing into contact, and/ordetection and/or measurement, of the method of the invention. Thus, themeasurement and/or the detection can readily be effected in wells ofmicrotitration plates. On such a support, for example the abovementionedstrip, the wells can be coated with antibody, for example goat or rabbitantibodies, which are directed against the mouse anti-estradiolantibodies. The silylated estradiol can also be bound to the bottom ofthe wells.

As a result of the present invention, the inventors have succeeded indeveloping a practical and rapid test for detecting HF at concentrationswhich can be of the order of 0.001 μg/ml in solution.

The sensitivity of the method of the invention advantageously makes itpossible to envisage any industrial application and, in particular, themeasurement of atmospheric hydrofluoric acid, which is the most useful.In this case, 1×10⁻² l of HF/10⁶ l of air (10 ppb) can easily bedetected. The performance of this method is therefore superior to thatof the field assays of the prior art, which were limited to detecting HFat concentrations of the order of some hundreds of ppb.

In general, the present invention makes it possible to obtain a resultin approximately ½ to 4 hours, depending on the nature of the sample andthe detection means employed. Even if some of the times taken are longerthan those for certain methods of the prior art, the method of theinvention is generally more sensitive.

The method of the invention advantageously applies to any samplescontaining hydrofluoric acid or fluoride ions: atmospheric sample,foodstuffs, plants and biological media. In each case, the conditionsfor sampling and for preparing a sample are the same as those of anyother method for assaying hydrofluoric acid or fluoride ions.

Another advantage of the present invention is that it is simple to carryout. For example, the technology can be identical to that ofimmunoassays: employment of ready-to-use reagents, easy-to-handledispensing, compactness, easy transport and relocation, possibility ofworking without the provision of energy, and reading with the naked eyeor by means of colorimetry using a simple pocket spectrophotometer.

Other applications and advantages of the present invention will alsobecome apparent from reading the description which follows and which isgiven by way of illustration, and in a non-limiting manner, withreference to the appended figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the part of the estradiol (Es) moleculewhich is recognized by its antibody (Ab). The attachment of an Si atomto the —OH of the part of the estradiol molecule recognized by theantibody prevents the antibody from recognizing this molecule.

FIG. 2 is a general scheme for detecting HF by means of the method ofthe invention in accordance with the first embodiment, in which thedetection is effected by competition.

FIG. 3 is a diagram of a competitive immunoassay (on the left) and ofthe measurement of the signal which is obtained using such an assay (onthe right). In this figure, S(%) represents the signal emitted by thetracer (T) which is bound to the antibody (Ab); and [Es] (M) representsthe molar concentration (mol/l) of the native estradiol.

FIG. 4 is a graph which shows the effect of different organic acids onthe desilylation of trimethylsilylestradiol which is obtained usingBTSFA (B/Bo is a measure of the signal in the form of a ratio betweenthe signal in the presence of HF (B for bound) and the signal in theabsence of HF (Bo for unbound) in an immunological test in accordancewith the protocol of Example 2 below. [H⁺] (M) represents the molarconcentration of acid for each acid identified in this graph.

FIG. 5 is a graph which shows a study of the conditions for desilylatingestradiol: temperature, and pH of the 50 mM phosphate (P) buffer (theB/Bo signal is measured as in FIG. 4).

FIG. 6 is a graph which shows the effect of different acids on thedesilylation of silylated estradiol in 50 mM phosphate buffer, pH 5.5.[H⁺] (M) represents the molar concentration of acid for each acididentified in this graph. The data are similar with hydrochloric acid,nitric acid or sulfuric acid (the B/Bo signal is measured as in FIG. 4).

FIG. 7 is a graph which shows the specificity of the detection of HF, ascompared to other nucleophiles and halogens, by the method of theinvention. [P] (M) represents the molar concentration of halogen orother nucleophiles for each halogen identified in this graph (the B/Bosignal is measured as in FIG. 4).

FIG. 8 is a graph which shows the specificity of the detection of HF bythe method of the invention in the presence of different salts [X⁺F⁻](M) represents the molar concentration of fluorine salt for each saltidentified in this graph (the B/Bo signal is measured as in FIG. 4).

FIG. 9 is a graph which shows the correlation between the concentrationof fluorine [F] (in μmol/l) which is detected (d) by the method of theinvention and the theoretical concentration ([F]_(th)) in samples ofatmosphere which is contaminated with fluorine.

FIGS. 10 to 14 are graphic representations of experimental resultsobtained in regard to the recognition of unsilylated estradiol byspecific antibodies in accordance with non-competitive immunologicalassay protocols. In these figures:

FIG. 10 depicts the signal (S) (mOD) obtained for the coupling ofestradiol carboxymethyl ether (EstCME) to microtitration platespossessing amino groups (aminated Nunc plates) in dependence on theconcentration of the estradiol (in μg/ml).

FIGS. 11 and 12 depict the signal (S) (mOD) (absorbance measured at 414nm) obtained for the coupling of estradiol carboxymethyl ether topolylysine (poly-Lys)-activated microtitration plates in dependence onthe concentration of polylysine (in μg/ml).

FIG. 13 depicts a test of the silylation of estradiol carboxymethylether by MTSBTFA: the efficacy of the silylation is measured by theincrease in the signal (S) (mOD), in dependence on the dilution of theMTSBTFA (1/5; 1/2; 1/1) and on the temperature in degrees Celsius (°C.).

FIG. 14 depicts an immunometric format test following silylation of theestradiol carboxymethyl ether: the signal (S) (mOD) is measured independence on the presence or absence of HF. In this figure Borepresents the signal in the absence of HF (control).

FIG. 15 depicts, in the form of a graph, experimental results obtainedfrom implementing the method of the invention using a silylated organiccompound which is a tetrapeptide, AcSDKP: the signal (S(mOD)) ismeasured at 414 nm. Two series of assays were carried out: one seriesusing the tetrapeptide which was silylated with a first silylatingreagent (MTSBTFA), and one series using the tetrapeptide which wassilylated with a second silylating reagent (BSTFA). Three assays werecarried out in each series: at 22° C., at 37° C. and at 70° C., andmeasurements were made at 10, 30 and 60 minutes in each case.

FIG. 16 is a graphic depiction of experimental results which wereobtained in a study of the efficacy of the detection of the fluorine bythe method of the present invention in dependence on the desilylationtemperature (T(° C.)): the signal (S(mOD)) is measured at 414 nm. Thesilylated compound employed is that of FIG. 15 obtained with MTBSTFA.

FIG. 17 depicts, in the form of a graph, experimental results obtainedfrom implementing the method of the invention using a silylated organiccompound which is a derivative of estradiol: the signal (S(mOD)) ismeasured at 414 nm in dependence on the concentration of fluorine [F] inmicromoles/l (μM).

FIG. 18 diagrammatically depicts the mechanism of a non-competitiveimmunological detection as can be used in the detection step of themethod of the invention.

FIG. 19 is a graph which shows the results of detecting the fluorine bythe method of the invention in the presence of dimethyl sulfoxide(DMSO): B/Bo is a measure of the signal in the form of a ratio betweenthe signal in the presence of HF (B) and the signal in the absence of HF(Bo), and [F⁻] (M) is the molar concentration of fluoride ions.

EXAMPLES

The different aspects of the method of the invention has been studiedbelow: the setting-up of the immunological assay for estradiol, tests ofthe conditions for modifying the estradiol by silylation, verificationof the silylated organic compounds by mass spectrometry, testing of thereaction of the silylated compounds with HF, optimization of thechemical nature of the silylated derivatives, optimization of theconcentrations of reagents, sensitization of the measurement,optimization of the specificity of the measurement in regard to acids,testing of the applicability of the measurement. The main results ofthese studies are set out below.

The tests were carried out on samples of atmospheric hydrofluoric acid(simulation in the laboratory) and on samples of water containingfluoride ions.

In order to determine the power of the method of the invention, theinventors defined its sensitivity parameters. Its sensitivity representsthe quantity of HF which is required in order to engender a signal whichis statistically different from the signal which is obtained in theabsence of HF. In accordance with the method of the invention, thequantity of native (unsilylated) estradiol which is present in thesample to be measured depends on the quantity of fluorine which is toconvert undetectable silylated estradiol into detectable unsilylatedestradiol. For the purpose of measuring HF, therefore, the sensitivitywas defined as the requisite quantity of fluorine which, when broughtinto the presence of dimethyl tert-butyl estradiol, is able to induce asignficant decrease in the bond between the antibody and the enzymictracer.

Example 1 Application of the Method of the Invention to the Detectionand Measurement of the Fluorine by Competitive Immunoassay

In this example, the organic compounds which are selected are compoundswhich are derivatives of β17-estradiol.

1) Preparing the Anti-Estradiol Antibody

The antibody which was used in the assay was produced, in particular,from compounds which were derived from β17-estradiol and which werecoupled to bovine albumin with the aim of obtaining antibodies in mice.

The technique employed is that described in reference [39].

2) Preparing an Antibody Mouse Antibody from the Anti-Estradiol Antibody

Goat anti-mouse antibody antibodies marketed by the Immunotech company(Lumigny) are used for coating (dilution at which used: 5 μg/ml) 96-wellpolystyrene microtitration plates (Nunc). After having been coated, theplates are stored at 4° C. in a buffer containing 0.5% albumin.

Binding to the plate is effected by simple absorption. The incubationshould be sufficiently long to enable the protein to become attached.This preparation forms part of the general knowledge of the skilledperson.

3) Preparing the Enzymic Tracer

The enzymic tracer was obtained by coupling estradiol to acetylcholineesterase (AChE). Thiol groups are introduced into the estradiol usingN-succinimidyl S-acetylthioacetate. The estradiol which has thus beenmodified is coupled to the AChE, into which maleimide groups which reactwith the estradiol thiol groups have been introduced. The tracer waspurified by gel filtration and then stored in aliquot form at −20° C.

The documents with the reference numbers [44] and [45] describe thetechniques and the enzymic tracers which are used for this preparation,and which can be used generally in the present invention.

4) Products and Other Reagents

Estradiol was obtained from Sigma Aldrich. The silylating agents wereobtained from the Perbio company.

Hydrofluoric acid is obtained from the Merck-Eurolab company.

All the reagents, i.e. tracer, antibody, estradiol and samples, wereused while being diluted in an 0.05M phosphate buffer, pH 7, containingazide (0.01% w/v) (% w/v=ratio of weight in g to a volume of 100 ml)(that is, in this case, 0.01 g/100 ml), bovine alumin (0.5% w/v) andNaCl (0.9% w/v).

5) Optimization of the Silylated Estradiol Concentration Employed

The concentration of silylated estradiol brought into contact with thesample containing HF was optimized in order to enable desilylatedestradiol, and therefore the fluorine, to be detected subsequently.

Thus, if the concentration of silylated estradiol employed is very low,this low concentration does not always enable detection to be effectedwith great precision even if all the silylated estradiol is transformedinto estradiol.

A number of tests were carried out in order to determine the optimumconcentration of modified estradiol to be used for the desilylation inthe method of the invention. This concentration naturally varies independence on the reagents employed and on the operational conditions.

In the examples which are presented here, the concentration ispreferably 200 ng/ml.

It is possible to estimate that this concentration will in general, inthe case of estradiol and its derivatives, be from 1 to 2000 ng/ml,preferably from 2 to 500 ng/ml, with these ranges being given only as anindication, particularly for the conditions of the example.

6) Silylating the Estradiol

One volume of estradiol (2 mg/ml) is mixed with 4 volumes of MTBSTFA andthe whole is incubated at room temperature for from 30 minutes to 1hour.

The silylating agents employed are BTSFA or MTBSTFA. The products whichcan be obtained are depicted above.

Verification by liquid chromatography and mass spectrometry showed thatthe estradiol had been silylated.

Having once been silylated, the estradiol is diluted by a factor of 1000in 100% dimethylformamide (DMF), or else in 100% dimethyl sulfoxide(DMSO), before use.

7) Desilylating the Estradiol

2.5 μl of 1M phosphate buffer (pH=5.5) and 47.5 μl of sample, or of anHF solution of known concentration (standard series), are added to 50 μlof silylated estradiol.

The mixture is shaken and then left at 55-66° C. for 1 hour to drynessin a water bath.

The desilylation reaction can be stopped by diluting the mixture in anassay buffer (dilution by a factor of 40) which is 0.05 M phosphatebuffer, pH 7, containing azide (0.01% w/v) (% w/v=ratio of weight in gto a volume of 100 ml) (that is, in this case, 0.01 g/100 ml), bovinealbumin (0.5% w/v) and NaCl (0.9% w/v).

8) Enzymic Indicator: Measuring the Enzymic Activity of AcetylcholineEsterase (Ellman Reaction)

The method uses a pseudosubstrate, i.e. acetylthiocholine (AcTCh), whichis hydrolyzed at the same rate as acetylcholine. This hydrolysis leadsto the formation of thiocholine (TCh), which is able to reducedithionitrobenzene (DTNB). The reduced DTNB strongly absorbs in thevisible range (ε_(m 412 nm)=13600 mol⁻¹.cm⁻¹.l), producing a yellowcolor.

The AcTCh is used at a concentration at which the activity of the AChEis maximal, taking into account its inhibition by excess substrate. Inaddition, the quantity of DTNB employed is less than that of AcTCh so asto avoid colorimetric measurements after all the substrate has beentransformed. By contrast, the quantity of DTNB employed is in excess ascompared to the quantity of TCh produced so as to ensure that thehydrolysis of one molecule of AcTCh leads to the formation of only onemolecule of reduced DTNB.

The skilled person knows how to carry out the Ellman reaction and can,on the basis of the information which is provided here, withoutdifficulty find the operational conditions which are optimal forimplementing the invention.

9) Immunoassaying the Native Estradiol: Quantifying HF

The estradiol extracts which have previously been obtained are diluted1/40 and portioned out on assay plates in the presence of the tracer andthe anti-estradiol antibodies. The reaction volume is 200 μl.

The immunological reaction is carried out at ambient temperature for onehour.

The assay plates are washed in order to remove unbound tracer and 0.2 mlvolumes of indicator solution are added.

After approximately 1 h, the yellow color appears and optical densitymeasurements are carried out using a spectrophotometer.

10) Interpreting the Results

Using a standard HF series, it was possible to determine theconcentration of the fluorine contained in the samples in the assayswhich were performed, thus demonstrating the feasibility of the methodof the invention.

Example 2 Study of the Difference in the Specificity of HF Detection inthe Case of MTBSTFA-Modified Estradiol and in the Case of BTSFA-ModifiedEstradiol

In all the manipulations described below, the silylated estradiol wasused, during the desilylation, at a concentration which was the same forall the samples in a given experiment. This concentration was 200 ng/ml.

In the case of the other reagents, the concentrations were as follows:

-   -   mouse anti-estradiol antibody: 5 μg/ml;    -   acetylthiocholine: 7×10⁻⁴ M;    -   5-5′-dithiobisnitrobenzoate: 7×10⁻⁴ M; and    -   estradiol coupled to acetylthiocholine: approximately 1 ng/ml.

The tests were carried out in 300-μl wells on polystyrene strips in themanner explained in Example 1.

A) The estradiol which is silylated with BTSFA (which transfers atrimethylsilyl group) is cleaved (desilylation reaction) with HF,thereby recovering its native form. Such a cleavage can also be observedwhen using other acids: hydrochloric acid (HCl), nitric acid (HNO₃),sulfuric acid (H₂SO₄) and phoshoric acid (H₂PO₄). The results arepresented in the appended FIG. 4.

Therefore, when this silyl group is used, the desilylation reaction isnot always specific for the fluorine. This silylated estradiol istherefore suitable, instead, for detecting HF when the latter is on itsown or else for detecting HF when the other acids are not present.

B) Although exhibiting a lower solubility in aqueous medium, theestradiol which is silylated with MTBSTFA (which transfers a silyl groupwhich is more hydrophobic than that transferred by BTSFA) is cleaved(desilylation reaction) specifically by HF, thereby recovering itsnative form. Thus, as the appended FIG. 6, which was produced under thesame conditions, shows, the other acids which were tested do not cleaveestradiol which has been modified in this way (see Example 3 below).

The inventors therefore selected MTBSTFA for silylating the estradiol inthe examples which follow.

Example 3 Seeking Buffers which are Suitable for the FluorineDesilylation Reaction in the Method of the Invention

Since some acid entities also have an effect on the desilylation ofestradiol which has been modified using MTBSTFA (see Example 2), theinventors decided to seek buffer solutions which accentuated thespecific effect of the fluorine on the desilylation reaction andinhibited the non-specific effect due to the acid nature of the othercompounds which might be present.

When present at high concentrations (necessary for buffering the highacid concentrations), bases such as sodium hydroxide (NaOH) andpotassium hydroxide (KOH) also induce desilylation of the estradiol inthe absence of acid (data not shown). They cannot, therefore, be used.

Phosphate buffer (KH₂PO₄/K₂HPO₄) solutions which were 50 mM in phosphateion were also tested. They gave good results. The pH of these solutionswas studied in order to ensure that their acidity did not result in thesilylated estradiol being spontaneously desilylated.

The method of the invention can therefore advantageously be carried outin a buffered medium, for example when the sample may contain otheracids or bases which are able to induce spontaneous desilylation of theorganic compound and therefore reduce the detection and/or measurementsensitivity of the method of the invention.

Furthermore, the inventors observed that, under certain conditions, adesilylation temperature which was too high could result in the modifiedestradiol being spontaneously desilylated (data not shown). For thisreason, each of the different buffering agents was studied at differenttemperatures.

The appended FIG. 5 shows the results which were obtained. In thisfigure “E2M” represents dimethylsilyl tert-butyl estradiol and “A.T.”represents the ambient temperature.

While, in the case of phosphate buffer solutions at pH 5.5 or 6,spontaneous desilylation of the silylated estradiol occurs at 94° C.,there is no such desilylation at 54° C.

In conclusion, the preferred conditions for desilylating silylatedestradiol were set as follows: temperature between 54 and 64° C. in a 50mM phosphate buffer at pH 5.5. As will be understood, these conditionswere determined for estradiol and the abovementioned silyl group, andunder the previously mentioned operational conditions.

On the basis of the information which has been provided herein, theskilled person will readily be able to determine other conditions independence on the silylated organic compound selected, on the sample andon the conditions under which the method of the invention isimplemented.

Example 4 Effect of Different Acids on the Implementation of the Methodof the Invention

The effects of various other acids on the desilylation were tested underthe conditions mentioned above in Example 3.

The results of these tests are depicted in FIG. 6. In this figure,“F.ac” represents formic acid; “ac.ac” represents acetic acid; “P.ac”represents phosphoric acid; “OP.ac” represents orthophosphoric acid; and“T.ac” represents trifluoroacetic acid.

These results clearly show that acids other than HF have almost noeffect on estradiol which is silylated by MTBSTFA.

The silyl group which is selected therefore plays a role in thespecificity of the detection of the fluorine. This fact is to beconsidered when chemical components other than the fluorine are presentin the sample being tested, especially if these components are able toinduce desilylation of the organic compound being employed.

Example 5 Specificity for Fluorine as Compared to Other Halogens andNucleophiles

Fluorine is a halogen and also has a nucleophilic character. In order totest the specificity for fluorine in the estradiol desilylationreaction, the effect of other halogens, such as Bu₄NBr, Bu₄NCl, Bu₄NIand KI, and of nucleophiles, such as 4-nitrophenol and 4-nitroimidazole,was studied under the operational conditions of the previous examplesand with the estradiol being silylated with MTBSTFA.

The appended FIG. 7 depicts the experimental results which were obtainedin this example and demonstrates the specific effect of fluorine ascompared with other products.

It clearly appears that, in comparison with the other products tested,fluroine is very specific in the estradiol desilylation reaction.

Example 6 Cleavage by HF and by Fluorine Ions

In this example, various fluorine salts were brought into contact withMTBSTFA-silylated estradiol under the experimental conditions used inthe preceding examples.

The appended FIG. 8 collates the results which were obtained. In thisfigure, the usual chemical symbols were used to identify the differentfluorine salts which were tested.

The fluorine ions behave similarly to HF in regard to cleaving thesilylated estradiol, with this confirming the specificity of thereaction for fluorine.

Example 7 Analytical Power of the Method of the Invention, and Effect ofthe Addition of an Organic Solvent

Under the conditions described in the above examples, the sensitivityfor measuring HF is approximately 5×10⁻⁵ M (that is 1 μg/ml) in the caseof HF which is diluted in aqueous medium, as FIGS. 7 and 8 show.

During the course of supplementary studies, the inventors noted that thesensitivity of the method of the invention can be increased stillfurther by adding a water-miscible organic solvent to the aqueousdesilylation solution.

The experiment was carried out using dimethylformamide (DMF) (seesilylation of estradiol) and dimethyl sulfoxide (DMSO).

The sensitivity can be improved by a factor of 500 (approximately 10⁻⁷M, that is 0.001 μg/ml) by using dimethyl sulfoxide (DMSO) as solventfor implementing the desilylation in accordance with the method of theinvention.

FIG. 19 depicts the dose-response curve for fluoride ions in the case ofa reaction carried out in the presence of dimethyl sulfoxide at aconcentration of 95% by volume based on the total volume of thecontacting aqueous solution. These results are to be compared with thosefrom the previous examples.

The addition of the organic solvent to said contacting aqueous solutiontherefore substantially increases the sensitivity for detecting and/ormeasuring fluorine by the method of the invention.

Example 8 Using the Method of the Invention when the Compound of theInvention is an Estradiol Derivative

In this example, the organic compound employed for implementing themethod of the invention is an O-methyl derivative of estradiol (E2OCH₃):17-betaestradiol 3-methyl ether of the formula:

The operational conditions which are used are those explained inExamples 1 and 2 above.

The experimental results which are obtained are reported in the appendedFIG. 17. It is indeed observed that E2OCH₃ is desilylated in thepresence of fluorine.

There are, therefore, cogent reasons for using an estradiol derivativein the method of the invention.

Example 9 Using the Method of the Invention when the Organic Compound isa Peptide Possessing Hydroxyl Functions

In this example, the organic compound is a peptide. The peptide is atetrapeptide, i.e. acetylated Ser-Asp-Lys-Pro (AcSDKP) of the formula:

AcSDKP is a peptide which possesses hydroxyl groups, advantageouslyallowing it, at one and the same time, to be silylated and to beattached to a solid phase by its carboxyl or amino groups.

The silylation reactions of this peptide were studied first of all, withthis being followed by a study of the use of this silylated compound inthe method of the invention.

AcSDKP (1 mg/ml) was silylated with undiluted BSTFA or MTBSTFA for 10,30 or 60 minutes at 22° C., 37° C. or 60° C. After the reaction, thesilylated AcSDKP was detected by an AcSDKP-specific competitive assay bymeans of the technique described in document [5] and using rabbitpolyclonal antibodies. In this case, an increase in the signal indicatesthat immunoreactivity has been lost and that the silylation hastherefore been effective.

As the appended FIG. 15, which reports the experimental resultsobtained, shows, MTSBTFA decreases the reactivity of the AcSDKP at 60°C., with this suggesting that the compound has been silylated.

Desilylation tests were then carried out on this silylated peptide inthe presence of HF, in accordance with the present invention. Theappended FIG. 16 collates the results which were obtained. It is seenthat the HF desilylates the peptide. Thus, the loss of the silylatedgroup results in immunoreactivity being increased and therefore in thesignal becoming weaker. As the figure shows, the signal decreases in thepresence of HF.

The method of the invention can therefore be implemented when using ahydroxylated peptide as the organic compound.

Example 10 Detecting and Measuring Fluorine in Solution

In order to verify the validity of the method of the invention, theauthors tested various mineral waters whose concentration of fluorine isknown.

The protocol employed is that of Example 1 and the organic compoundselected is estradiol silylated with MTBSTFA (dimethyl tert-butylestradiol).

Table I below collates the results which were obtained in this example.The table shows that the presence of fluorine was detected in everycase.

The correlation between the theoretical concentration and theconcentration which was found appears correct in the case of the mostconcentrated water samples. It is possible that the nature of thefluorine present in the sample is the cause of the few variations whichwere observed.

TABLE I Measurement of fluorides in mineral waters [dry [F] Water[fluorides] extracts] detected % of F samples mg/ml μM mg/ml pH μM foundVichy St 9 470 4774 6.6 780 ± 271 170 ± 60  Yorre San- 0.61 32 1074 7.5151 470 Pellegrino Badoit 1 52 1200 6 28.5 ± 12   50 ± 20 Vichy 6 1353325 6.8 305 ± 81  97 ± 26 Célestin Quézac 2.1 100 — —  53  53

Example 11 Detecting and Measuring Fluorine from Gaseous Media

The HF to be measured is frequently in gaseous form and it is necessaryto pass it into aqueous solution in order to measure it. Since they didnot have any air which was contaminated with HF, the inventorsartificially created such samples by evaporating an HF solution of knownconcentration in a sealed container. Once the HF had evaporated, the airwas pumped into, and caused to bubble in, a collecting buffer (DMF).

Using a system which enables 100 liters of air, whose concentration ofHF is 1.2×10⁻² l/10⁶ l of air (12 ppb), to be filtered, an HFconcentration of 10⁻⁵ M is obtained in solution (subject to a 100%yield).

The protocol which is used for detecting the fluorine is that describedin Example 1. The organic compound selected is estradiol silylated withMTBSTFA (dimethyl tert-butyl estradiol).

The results which are presented in FIG. 9 show that the fluorine isdetected in every case. The correlation between the theoretical anddetected concentrations of fluorine is fairly good.

The method, according to the invention, for detecting HF in airtherefore easily makes it possible to detect HF at concentrations of theorder of 10⁻² l of HF/10⁶ l of air (10 ppb).

Example 12 Applying the Method of the Invention to the Detection andMeasurement of Fluorine by Means of Non-Competitive Immunoassay

1) General Strategy

The aim is to use the previously acquired results, namely thepossibility of modifying the immunological recognition of a silylatedcompound by the action of HF.

As compared to the previous assay format (competitive), the immunometricapproach consists in immobilizing the silylated compound covalently on asolid surface. Its transformation by HF leads to an immunoreactive form,and therefore to the appearance of a signal, contrary to the competitiveassay in which the signal disappears.

This format frequently results in superior sensitivity, probably due tofavorable thermodynamics linked to the reagents which are in excess andto easier reading, based on the fact that it is easier to see or measurethe appearance of a signal than its disappearance.

2) Reagents and Protocol

The antibody employed is that described in reference [39].

The biotinylation of the rabbit antibody and the labeling of thestreptavidin or the antibodies with acetylcholine esterase were carriedout in the laboratory using the customary techniques known to theskilled person.

All the chemical reagents and products were obtained from Sigma orMerck. The compounds obtained come from Sigma or Steraloids (in the caseof the estradiol derivatives).

The assay buffer is an 0.05 M phosphate buffer, pH 7.4, containing azide(0.01%), bovine albumin (0.5%) and NaCl (0.9%).

The enzymic indicator consists of a mixture of acetylthiocholine anddinitrifluorobenzene. Following reaction with the tracer, it produces ayellow color which is visible to the naked eye or which can bequantified using a spectrophotometer at 414 nm.

The plates which were used for carrying out the experiments, inparticular for attaching the estradiol derivative, come from Nunc(Rochester, USA).

The silylating agents employed are BSTFA and MTBSTFA.

The estradiol which is used is shown below. The advantage of thiscompound is that it makes it possible to use antibodies for thedetection and the basic principle which was previously developed for thecompetitive assay.

The inventors firstly verified that the anti-estradiol antibodies whichwere obtained recognized the unsilylated compound (53% cross-reactionwith the antibodies as compared to estradiol) and not the silylatedcompound. The verification was positive.

Structure of Estradiol Carboxymethyl Ether (EstCME)

The inventors then tested the covalent attachment (binding) of theestradiol to two types of plate: plates possessing amino groups(Nunc-NH₂ plates) and plates which were activated with polylysine(poly-Lys).

3) Tests of the Attachment of the Estradiol Derivative

The EstCME was incubated on Nunc plates in the presence of equimolarconcentrations of N-hydroxysuccinimide (NHS), an agent which binds theamino groups on the plate to the carboxyl groups of the antigen, at 22°C. for 3 hours. Visualization was effected using a 100 ng/mlanti-estradiol antibody at 20° C. for 4 hours and then using a goatanti-rabbit antibody antibody coupled to acetylcholine esterase (2Ellman units of gAb-AchE/ml) at 22° C. for 2 hours. The enzymic activityis visualized by adding the Ellman reagent, a substrate of the AchE.

The results which were obtained are reported in the appended FIG. 10.They show that the estradiol is bound effectively and that the maximumappears to be achieved at the highest concentration tested (100 μl/ml).

This initial experiment was repeated using plates on which polylysine(poly-Lys) (between 1 and 100 μg/ml in phosphate buffer) had previouslybeen absorbed.

The results which were obtained are reported in the appended FIG. 11. Inthis case, the results are of the same order whatever the concentrationof polylysine studied. This furthermore demonstrates that the aminogroups carried by the polylysine are more accessible for binding thechosen estradiol.

In order to study the influence of the concentration of polylysine inmore detail, the test was repeated using a wider range of polylysineconcentrations. The results which were obtained are reported in thegraph in the appended FIG. 12. They show an optimum for polylysinearound 1 μg/ml.

The two preceding figures (FIGS. 11 and 12) appear to indicate effectivecoupling of the estradiol derivative to the plates, with a plateauobtained at a polylysine concentration of 1 μg/ml and an estradiolcarboxymethyl ether concentration of 10 μg/ml.

4) Silylating the Estradiol Derivative

In addition, the inventors sought to demonstrate the best conditions forsilylating the above mentioned compound. The estradiol carboxymethylether was reacted, for from 0 to 60 minutes, with MTBSTFA at variousconcentrations (1/5, 1/2 and 1/1) and two temperatures (22° or 37° C.).The silylation is measured by means of the competitive assay of thesilylated estradiol in which the silylated estradiol loses itsrecognition (and in which the signal should increase).

The results which were obtained are reported in the appended FIG. 13.This figure shows that, under conditions of high MTBSFA concentration,the estradiol carboxymethyl ether loses its immunoreactivity. This lossis indicated by an increase in the signal, due to the absence ofrecognition. The MTBSFA is demonstrated to have a dose effect.

These results taken together, i.e. efficacy of the coupling to the solidphase and of the silylation, enabled the following coupling/desilylationtest to be carried out on plates.

5) Coupling/Desilylation Test

The estradiol carboxymethyl ether (8.3 mg/ml) was reacted, at 22° C. for1 hour, in the presence of MTBSTFA at the same concentration and thendiluted in water to 1 μg/ml and reacted, at 22° C. for 1 hour and in thepresence of NHS, on plates which were coated with polylysine (1 μg/ml).

Desilylation was effected, at 22, 37 or 64° C. for 1 hour, in theabsence or presence of HF at a concentration of 1 mM.

The results are reported in the appended FIG. 14. As this figure shows,it is possible to observe an increase in the immunoreactivity whichcorresponds to a desilylation of the silylated estradiol carboxymethylether in the presence of HF.

This thereby demonstrates the detection of HF by the method of theinvention when the silylated organic compound is bound to a support.

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1. A method for detecting and/or measuring the concentration of fluoride(F⁻) or hydrogen fluoride (HF) in a sample, comprising contacting, inaqueous solution, said sample with a silylated organic compound in orderto obtain a measurement solution, wherein said silylated organiccompound being desilylated when it is in the presence of hydrofluoricacid or of fluoride, and wherein the silylated organic compound and thedesilylated organic compound being able to be detected and/or measuredseparately from each other; and detecting and/or measuring, in saidmeasurement solution, the appearance of the desilylated organiccompound, or the disappearance of the silylated organic compound, whichtakes place if fluoride or hydrogen fluoride is present in the sample,wherein said silylated organic compound is:

in which R¹, R² and R³ are independently selected from C₁ to C₆ alkylsand R⁴ is an organic compound.
 2. The method as claimed in claim 1, inwhich R¹, R² and R³ are independently selected from the group consistingof methyl, ethyl, propyl and butyl.
 3. The method as claimed in claim 1,in which the organic compound is a hydroxylated compound having amolecular weight of from 250 to 200,000 g.mol⁻¹.
 4. The method asclaimed in claim 1, in which the organic compound is a hydroxylatedcompound selected from the group consisting of estradiol, peptides,homovanillic acid, amphotericin, steroids, cytokines, arachidonic acidand derivatives thereof.
 5. The method as claimed in claim 1, in whichthe detection and/or measurement, in said measurement solution, of theappearance of the desilylated organic compound, or of the disappearanceof the silylated organic compound, is carried out by means of gaschromatography.
 6. The method as claimed in claim 1, in which thedetection and/or the measurement, in said measurement solution, of theappearance of the desilylated organic compound, or of the disappearanceof the silylated organic compound, is carried out by means of animmunological test using one or more antibodies which is/are directedeither against the desilylated organic compound or the silylated organiccompound.
 7. The method as claimed in claim 6, in which theantibody(ies) is/are (a) monoclonal antibody(ies).
 8. The method asclaimed in claim 6, in which the immunological test is acompetitive-type or non-competitive-type immunoassay.
 9. The method asclaimed in claim 1, in which the organic compound is estradiol or one ofits derivatives.
 10. The method as claimed in claim 1, in which theorganic compound is selected from the group consisting ofestra-1,3,5-triene-3,17 μ or 17 μ-diol, and their derivatives.
 11. Themethod as claimed in claim 1, in which the silylated organic compound isused at a concentration of from 1 to 2000 ng/ml in the contacting step.12. The method as claimed in claim 1, in which the aqueous solution isbuffered to pH 4.5 to 5.5.
 13. The method as claimed in claim 1, inwhich the contacting is effected at a temperature of from 54 to 64° C.